Method of enzymatically measuring glycated protein

ABSTRACT

A sample containing a glycated protein is treated with Protease XIV or a protease from Aspergillus genus, thereafter (or while treating the sample with the above protease) FAOD (fructosyl amino acid oxidase) is caused to react with the sample so as to measure the amount of oxygen consumed by the FAOD reaction or the amount of the resultant reaction product, thereby to measure the glycated protein. 
     According to the above method, the glycated protein can be fragmented while the decomposition of the FAOD itself is prevented, thereby to facilitate the binding of the protein with the FAOD and to improve the sensitivity of the detection.

TECHNICAL FIELD

The present invention relates to a method of measuring a glycatedprotein by using fructosyl amino acid oxidase (hereinafter, referred toas "FAOD"). More particularly, the present invention relates to a methodwhich is capable of measuring a glycated protein with greater accuracyand high sensitivity, and is easily applicable to clinical examination(or clinical diagnostic test), etc., and also relates to a proteasewhich is suitably usable for such a measurement method.

BACKGROUND ART

A glycated protein is a substance which is produced by the non-enzymaticand irreversible binding of the amino group of an amino acidconstituting a protein, with the aldehyde group of a reducing sugar suchas aldose. Such a non-enzymatic and irreversible binding reaction isalso called "Amadori rearrangement," and therefore the above-mentionedglycated protein may also be called "Amadori compound" in some cases.

The rate of the formation of the glycated protein generally depends onthe concentration of the protein and the reducing sugar as raw materialsfor providing the above glycated protein, the time period of the contactbetween these raw materials, and the temperature at the time of theglycation reaction. As a matter of course, as the amount of the aboveprotein and reducing sugar is increased, as the time period of thecontact therebetween is increased, or as the temperature becomes higher(within a range such that the protein is not denatured), the rate of theformation of the glycated protein as a reaction product is increased,and the amount of the reaction product is also increased.

On the other hand, in a living organism (or "in vivo"), since theconcentration of the glycated protein is changed depending on the halflife of the protein as the raw material for the above glycationreaction, various kinds of information on the living organism can beobtained by measuring the concentration of the glycated protein.

Among the above-mentioned glycated proteins, for example, afructosylamine derivative produced by the glycation of hemoglobin inblood is called "glycohemoglobin", one produced by the glycation ofalbumin is called "glycoalbumin", and a derivative (having a reducingability) produced by the glycation of protein in blood is called"fructosamine".

Since the concentration of these glycated protein derivatives in bloodreflects the average concentration of blood sugar in a living organismfor a certain period of time in the past, the measured value of theconcentration of the above glycated protein derivative in blood may be asignificant indicator of the diagnosis of the symptom of diabetes and ofthe monitor or control of such a symptom. Accordingly, also from aclinical viewpoint, it is very useful to establish a method of measuringthe concentration of the glycated protein in blood.

Heretofore, it has been known that a glycated protein in a sample (orspecimen) can be measured, e.g., by causing an oxidoreductase to act onthe glycated protein and measuring the amount of the oxygen consumed inthis reaction or the amount of the product (such as hydrogen peroxide)based on the action of the oxidoreductase (e.g., Japanese PatentPublication (JP-B; "Kokoku") Hei-5-33997 (i.e., 33997/1993), JP-BHei-6-65300, Japanese Laid-Open Patent Applications (JP-A; "Kokai")Hei-2-195900, JP-A Hei-3-155780, JP-A Hei-4-4874, JP-A Hei-5-192193,JP-A Hei-6-46846, JP-A Hei-7-289253, JP-A Hei-8-154672 and JP-AHei-8-336386 may be referred to).

In addition, there is known a method of measuring a glycated protein forthe purpose of the diagnosis of diabetes (JP-A Hei-2-195899, JP-AHei-2-195900, JP-A Hei-5-192193 corr. to European Publication EP0526150A, JP-A Hei-6-46846 corr. to EP 0576838A, JP-A Hei-7-289253, JP-AHei-8-154672 and JP-A Hei-8-336386 may be referred to).

In general, an enzymatic reaction using a glycated protein as asubstrate is represented by the following formula.

    R.sup.1 --CO--CH.sub.2 --NH--R.sup.2 +O.sub.2 +H.sub.2 O→R.sup.1 --CO--CHO+R.sup.2 --NH.sub.2 +H.sub.2 O.sub.2

(wherein, R¹ represents the aldose residue of a reducing sugar and R²represents a residue of an amino acid, protein or peptide.)

As an enzyme catalyzing a reaction using the above glycated protein as asubstrate, FAODs (fructosyl amino acid oxidases) from various kinds ofmicroorganism are known. Our research group has already obtained FAODsfrom microorganisms belonging to Fusarium genus, Gibberella genus,Penicillium genera, etc., and has showed that these FAODs are useful formeasuring a glycated protein (JP-A Hei-7-289253, JP-A Hei-8-154672 andJP-A Hei-8-336386 may be referred to).

Among the above-mentioned various kinds of FAODs, the FAOD from Fusariumoxysporum S-1F4 (hereinafter, referred to as "FAOD-S") and the FAOD fromGibberella fujikuroi (hereinafter, referred to as "FAOD-G") have anactivity on fructosyl-lysine and/or fructosyl-polylysine, and thereforeit has been found that these enzymes are useful for measuring humanserum or human glycated albumin (JP-A Hei-7-289253).

Accordingly, it is expected that if a method of measuring a glycatedprotein by using these FAODs is established, the above-mentioned methodusing FAOD becomes applicable to a general-purpose examining apparatus,and such a measuring method can be effected with lower cost for ashorter period of time as compared with those in the conventionalmethods such as one using HPLC (high-performance liquid chromatography)and one using antibody. Further, in such a case, it becomes possible toaccurately measure the glycated protein in a component of a livingorganism by utilizing the specificity of the above FAOD enzyme, andtherefore the measurement of the glycated protein using the FAOD enzymeis fully expected for the mass screening examination in a medicalcheckup or a curative marker for diabetics.

In the measurement of a glycated protein using the FAOD, it is preferredthat the glycated protein as a substrate is efficiently bound to thesubstrate binding site of the FAOD as an enzyme (or catalyst).Accordingly, in order to enhance the rate of the enzymatic reaction, itis important to design the substrate so as to enhance the efficiency ofthe above binding. The reason for this is that the FAOD has a tendencysuch that it has a higher activity on a glycated peptide (having a lowermolecular weight than that of protein) than the activity on a glycatedprotein, and has a still higher activity on a glycated amino acid(having a still lower molecular weight than that of the peptide) thanthe activity on the glycated peptide.

With respect to the FAOD, it is well known that the reaction rate forthe above-mentioned FAOD is increased by converting a glycated proteinpresent in a living organism component into corresponding smallfragments (i.e., decreasing the molecular weight of the protein) by useof a protease. As described above, it is theoretically possible to use aprotease which completely digests or fragments the glycated protein intoamino acids because the glycated amino acids are most preferred in viewof the affinity of the substrate with the FAOD. However, such a methodhas a problem that it requires a considerably long period of time forthe fragmentation treatment of the protein into amino acids.Accordingly, it is preferred to use a protease which selectively cleavesthe glycated protein at the site of a glycated amino acid present in theprotein, in view of the balance between the affinity of the FAOD withthe substrate and the period of time required for the digestion orfragmentation.

However, there are many kinds of proteases, and the size or dimension ofthe substrate which is suitable for the FAOD enzymatic reaction may varydepending on the kind of the FAOD to be combined with the protease.Accordingly, in practice, preferred combinations of protease and theFAOD are considerably restricted.

In the above-mentioned technical field, it is known that various kindsof proteases are useful in combination with certain kinds of the FAODs,and those proteases are roughly classified into endo-type proteases andexo-type proteases.

The former, endo-type protease, is an enzyme which decomposes a proteinfrom the internal site thereof. Specific examples thereof includes:trypsin, α-chymotrypsin, subtilisin, proteinase K, papain, cathepsin B,pepsin, thermolysin, Protease XIV, protease XVII, protease XXI,lysyl-endopeptidase, prolether, bromelain F, etc.

On the other hand, the latter, exo-type protease is an enzyme whichsequentially decomposes a peptide chain from the end thereof. Specificexamples thereof includes: aminopeptidase, carboxypeptidase, etc.

JP-A Hei-5-192193 discloses, as proteases useful for measuring aglycated proteins, Proteinase K, pronase E, ananine, thermolysin,subtilisin, and cow pancreas proteases. Actually, the protease disclosedin JP-A Hei-5-192193 is subjected to the fragmentation treatment of aglycated protein present in a sample, and then is inactivated by theincubation at 55° C. for 30 minutes. The reason for the inactivation ofthe protease is to suppress or prevent the fragmentation of FAOD per seas a catalyst by the protease, in the course of the next step of thereaction between the glycated protein and the FAOD.

If the FAOD per seas a catalyst is fragmented, as a matter of course,there is decreased the amount of oxygen to be consumed or the amount ofhydrogen peroxide to be produced based on the action of the FAOD on theglycated protein, and as a result, the sensitivity for detecting theglycated protein is decreased. Further, since such fragmentation of theFAOD also has an effect on the accuracy in the results of measurement ofthe glycated protein per se, the above-mentioned inactivation treatmentof the protease has generally been considered to be an essentialtreatment, as long as the protease is used in combination with the FAOD.

However, it is not easy to select a protease satisfying theabove-mentioned requirement (i.e., preferable matching thereof with theFAOD) from known proteases. For example, JP-B Hei-5-33997 teaches noneof specific protease, and JP-A Hei-5-192193 only discloses proteases(protease K, protease E) to be used in combination with ketoamineoxidase which has been obtained from Debliomyces genus.

Thus, according to the present inventors' experiments, it has been foundthat, when a glycated protein present in a sample is actually treatedwith the protease disclosed in the above JP-A Hei-5-192193, then theprotease was inactivated by heating at 55° C. for 30 minutes, and theFAOD was reacted with the resultant product, the amount of hydrogenperoxide as a reaction product or the amount of oxygen consumed in thereaction is small, and as a result, the detection sensitivity isinsufficient.

Proteases can also be inactivated by the addition of an inhibitor, otherthan to the heat denaturation by heating. However, there are somecombinations of a protease and an inhibitor which cannot completelyinactivate the protease (i.e., wherein a certain degree of proteaseactivity still remains).

The above inactivation of the protease is based on the phenomenon thatthe inhibitor binds to the active center of the protease to which thesubstrate is to be bound. In order to cause the inhibitor to bind to theactive center of the protease, it is necessary to add the inhibitor tothe reaction system after the completion of the protease reaction, andthen to cause a reaction to occur at a certain temperature for a certainperiod of time after the addition of the inhibitor. Accordingly, theperiod of time required for the entire measurement of the glycatedprotein is increased by the period of time required for the inactivatingreaction of the protease.

An object of the present invention is to provide a method ofenzymatically measuring a glycated protein which can solve the problemsencountered in the prior art, and to provide an enzyme which ispreferably applicable to such a measurement method.

Another object of the present invention is to provide a method ofmeasuring a glycated protein with better accuracy and highersensitivity, and to provide an enzyme which is preferably applicable tosuch a measurement method.

DISCLOSURE OF INVENTION

As a result of earnest study, the present inventors have found that aspecific protease provides good matching with an FAOD which is suitablefor measuring a glycated protein (such as glycated albumin) in a livingorganism component in various aspects, whereby the glycated protein canbe measured with accuracy and high sensitivity, and that such acombination is very useful for achieving the above-mentioned object.

The protease according to the present invention is based on the abovediscovery, and is a protease to be used for measuring a glycated proteinin a sample in combination with FAOD (fructosyl amino acid oxidase).

The present invention also provides a method of measuring a glycatedprotein by causing FAOD to act on a sample containing a glycatedprotein, wherein the glycated protein is treated with a protease underan acid condition.

The present invention further provides a method of measuring a glycatedprotein by causing protease and FAOD to act on a sample containing aglycated protein, wherein a protease from Aspergillus genus is used asthe protease.

The present invention further provides a method of measuring a glycatedprotein by causing protease and FAOD to act on a sample containing aglycated protein, wherein Protease XIV is used as the protease.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing a relationship between the concentration ofglycated albumin and the resultant absorbance (glycated albuminmeasurement-albumin reagent) in Example appearing hereinafter.

FIG. 2 is a graph showing the pH-dependency of the relative activity ofa protease (optimum pH of A. melleus BP-6277 protease) in Example.

FIG. 3 is a graph showing the temperature dependency of the relativeactivity of a protease (temperature dependency of A. melleus BP-6277protease) in Example.

FIG. 4 is a graph showing the results of the measurement of glycationratios of HSA by use of a commercially available protease (trade name:Sumizyme MP) in combination with FAOD-S (measurement of the glycationratio with Sumizyme MP/FAOD-S) in Example.

FIG. 5 is a graph showing the results of measurement of glycation ratiosof HSA by use of the protease obtained in the example with the FAOD-S(measurement of the glycation ratios using A. melleus BP-6277/FAOD-S).

FIG. 6 is a graph showing the relative activity values of an acidprotease obtained in Example at various pH values, provided that theactivity value of the acid protease at pH 5 (optimum pH) is treated as"100" (reference).

FIG. 7 is a graph showing the relative activity values of an acidprotease obtained in Example at various temperatures, provided that theactivity value of the acid protease at 50° C. (optimum temperature) istreated as "100".

FIG. 8 is a graph showing the results of the absorbance measurementbased on the FAOD reaction with respect to various glycation ratios ofHSA (human serum albumin) in Example.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described in detail, withreference to the accompanying drawings as desired.

(Sample)

In the measurement method according to the present invention, it ispossible to use any sample (or specimen) containing a glycated protein(typically, a sample produced by or taken out from a living organism).Specific examples of the sample may include those from a living organismsuch as blood (whole blood, plasma or blood serum) and urine.

In general, the treatment of the above-mentioned sample with a proteasemay be conducted in accordance with an instruction manual available fromthe corresponding supplier. For example, it is preferred to incubate thesample with the protease in a Tris-HCl buffer (pH 8.0) for about 30minutes at 50° C. in the case of the Sumizyme MP (trade name; mfd. bySin Nippon Kagaku Kogyou Co. Ltd.) appearing hereinafter, or at 37° C.in the case of the Protease XIV (trade name; mfd. by Sigma Co.), as theabove-mentioned protease.

(Protease)

In the present invention, in view of provision of good detectionsensitivity, it is preferred to select the protease to digest orfragment a glycated protein in a sample, in accordance with an FAOD tobe used in combination therewith.

As the above-mentioned protease, it is possible to use one kind ofprotease, or plural kinds of proteases in a combination or in a mixturethereof. At this time, as desired, it is also possible to use pluralkinds of proteases so as to fragment a glycated protein morespecifically and to enhance the sensitivity of the detection.

In the present invention, Protease XIV (trade name; mfd. by Sigma Co.)or a protease from Aspergillus genus may preferably be used, in view ofeasiness in the accurate measurement of the glycated protein. As theprotease from Aspergillus genus, a protease from Aspergillus melleus(hereinafter, referred to as "A. melleus") may particularly preferablybe used.

Preferred examples of such a protease from A. melleus may include: aprotease from a specific A. melleus strain (name and address of thedepositary institution: National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry; 1-3, Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, 305-8566 JAPAN; date of deposition: Mar. 3, 1998;deposition number: FERM BP-6277). It has been confirmed by the presentinventors' investigation that this protease has substantially the sameenzymatic characteristics as those of the commercially availableprotease "Sumizyme MP" to be described below (the same degree ofmatching property with the FAOD, or the same degree of activity; asdescribed in Example 4 appearing hereinafter).

The above-mentioned protease from A. melleus includes those having anoptimum pH value in the basic range and those having an optimum pH valuein the acidic range. Both of these types of proteases are usable in thepresent invention.

Similarly, specific examples of the A. melleus-produced protease to bepreferably usable in the present invention may include: Sumizyme MP(trade name; mfd. by; SHIN NIHON CHEMICAL CO., JP) which is used in thefield of food processing. Sumizyme MP is used as an enzyme for use inthe food industry, and it is inexpensive and can be supplied in a largequantity. Accordingly, this enzyme is advantageously usable in view ofthe application thereof to clinical examination such as screening.

Generally, the site of a glycated protein at which glycation occurs isdetermined by the kind of the protein, more specifically, by the kindand position (or site) of amino acids constituting the protein. When theabove-mentioned protein is albumin, the position thereof which is mostliable to be glycated is the lysine at a 525th position counted from theN-terminal of the albumin, and in most of glycated albumin, the lysineat the 525th position counted from the N-terminal is glycated.Accordingly, if a protease which preferably fragments this glycatedlysine is used, the sensitivity of the detection would be improved. Theabove-mentioned "Protease XIV" or protease from Aspergillus genus suchas A. melleus (e.g., Sumizyme MP) may preferably be used also from sucha viewpoint.

Further, in the present invention, it is preferred to use a proteasewhich can easily be inactivated by using a simple treatment after theuse thereof in the protease treatment. Specific examples of the simpletreatment may include a pH change, heating, addition of an inhibitor,etc. (Protease having optimum pH in acidic range)

In the present invention, a protease having an optimum pH value in theacidic range (i.e., pH value less than 7.0) may also be used, as long asit has a characteristic such that it can effectively fragment orcleavage the glycated amino acid at the glycated site of the glycatedprotein, can increase the absolute amount of oxygen consumed in thereaction based on FAOD, or the absolute amount of the reaction product(e.g., hydrogen peroxide) produced thereby, and can enhance thesensitivity of the detection.

When such a protease having an optimum pH value in the acidic range isused, it is possible to inactivate the protease after the fragmentationof the glycated protein, by simply adjusting the pH value to the optimumpH value of the FAOD (in the basic pH range, e.g., pH=8). Accordingly,in such a case, after the inactivation of the protease, the operationfor the FAOD reaction can be extremely simplified, and the treatment canbe conducted rapidly. In the method of adjusting the pH value, anyoperation for inactivating the protease by heating denaturation oraddition of an inhibitor can be omitted.

Specific examples of such a protease having an optimum pH value in theacidic range may include a protease having its an optimum pH value inthe acidic range which can be isolated or purified from the proteasesfrom the above-mentioned A. melleus.

(Inactivation of protease)

In the measurement of the glycated protein according to the presentinvention, for example, it is possible to initiate the FAOD reactionafter the fragmentation treatment of the glycated protein, byinactivating the protease and adding the FAOD. It is preferred tosuppress or prevent the above-mentioned fragmentation treatment of theFAOD with the protease, by utilizing such protease inactivation.

(FAOD)

In the present invention, it is preferred to select an optimum FAOD inaccordance with the kind of the glycated protein as an analyte or targetfor the measurement, since the glycation site is determined by therelationship with a living organism component (e.g., kind and positionof amino acids constituting the protein).

Specific examples of the FAOD usable in the present invention mayinclude: those which can be induced by cultivating microorganism orbacteria belonging to Fusarium genus, Gibberella genus, Penicilliumgenus, Aspergillus genus, etc. in the presence of fructosyl lysineand/or fructosyl N.sup.α -Z-lysine. Such FAOD can be obtained, e.g., bythe method as disclosed in JP-A Hei-7-289253, JP-A Hei-8-154672, JP-AHei-8-336386, etc.

In the present invention, among the above FAODs, it is particularlypreferred to use the FAOD-S from Fusarium oxysporum S-1F4 describedabove or the FAOD-G from Gibberella fujikuroi AKU 3802 (JP-AHei-7-289253) in view the activity thereof on fructosyl lysine and/orfructosyl polylysine, as the sites in human serum or human glycatedalbumin which are easily glycated.

(Titer of FAOD)

The titer of the FAOD to be used in the present invention may preferablybe measured by the following method.

(1) Method of Measuring Hydrogen Peroxide as Reaction Product by Use ofColorimetry

A. Rate Method

100 μl of 100 mM fructosyl N.sup.α -Z-lysine (FZL)-45 mM4-aminoantipyrine-60 units/ml peroxidase solution and 100 μl of 60 mMphenol solution are mixed with 1 ml of 0.1 M Tris-HCl buffer (pH value8.0) and 50 μl of an enzyme solution (of FAOD the titer of which is tobe measured), and then distilled water is added to the resultant mixtureso as to provide a total volume of the mixture of 3.0 ml.

The resultant solution is incubated at 30° C. for 2 minutes, and then 50μl of 100 mM of fructosyl N.sup.α -Z-lysine (FZL) solution is addedthereto, and the resultant mixture is subjected to the measurement ofthe absorbance thereof at 505 nm with the elapse of time. The amount(micro-moles) of hydrogen peroxide produced per one minute is calculatedby use of the molar absorption coefficient of the quinone dye (5.16×10³M⁻¹ cm⁻¹) formed by this reaction, and the resultant value is regardedas an enzyme activity unit (U).

B. End-point Method

In the same manner as in the above "method A", the substrate (FZL) isadded and the incubation is conducted at 30° C. for 30 minutes, andthereafter the resultant mixture is subjected to the measurement of theabsorbance thereof at 505 nm. Then, the enzyme activity is calculated byuse of a calibration curve which has preliminarily been prepared.

(2) Method of Measuring Oxygen Absorption by Enzymatic Reaction

1 ml of 0.1 M Tris-HCl buffer (pH value 8.0) and 50 μl of an enzymesolution are mixed, and the total volume of the mixture is adjusted to3.0 ml by use of distilled water. The resultant mixture is transferredinto an oxygen electrode cell mfd. by Rank-Brothers Co.

The above-mentioned solution in the cell is stirred at 30° C. so thatthe temperature thereof and the oxygen dissolved therein assume anequilibrium state, and thereafter 100 μl of 50 mM FZL is added to theresultant solution and the absorption of oxygen is continuously measuredby use of a recorder so as to determine the initial rate thereof. By useof a standard curve, the amount of oxygen absorbed per one minute iscalculated and the resultant value is regarded as an enzyme unit.

(Method of measuring glycated protein)

In the method of measuring a glycated protein according to the presentinvention, it is preferred that, while a glycated protein in a sample istreated with the above-mentioned protease so as to provide a state ofthe glycated protein with which the FAOD is liable to react, or afterthe glycated protein is treated with a protease, the FAOD is reactedwith the resultant product so as to measure the amount of oxygenconsumed in the above FAOD-substrate reaction or the amount of thereaction product produced by the reaction.

The reaction of the glycated protein (or product produced by thetreatment thereof with protease) with the FAOD produces hydrogenperoxide and glucosone. Both of the hydrogen peroxide and glucosone canbe used as the FAOD reaction product which is an analyte to be measuredin the subsequent step. The method of measuring the FAOD reactionproduct is not particularly limited, but may be one which isappropriately selected from known methods.

The amount of the hydrogen peroxide can be quantitatively determined byuse of any method known in the above-mentioned technical field, such ascolorimetry method or color-developing method (e.g., a measurementmethod employing a chromogen which is capable of producing a dye orcoloring matter along with the decomposition thereof by a catalysthaving a peroxidase or peroxidase-like activity), a measurement methodusing an electrochemical technique (e.g., a method using a hydrogenperoxide electrode), a method of measuring the amount of aldehydeproduced from hydrogen peroxide in the presence of catalase and analcohol.

In the present invention, e.g., it is possible to quantitativelydetermine the glycated protein by use of a calibration curve which haspreliminarily been provided by using the above-mentioned measurementmethod and samples respectively containing known amounts of the glycatedprotein. At this time, it is preferred that the activity of the FAOD isconstant. As desired, a sample containing a living organism component,etc., may preferably be subjected to the measurement after the sample isdiluted with a buffer solution.

(Color-developing system)

As the color-developing system for the colorimetry method using hydrogenperoxide, it is possible to use a system which produces a coloringmatter through oxidation condensation between a coupler such as 4-aminoantipyrine (4AA) and 3-methyl-2-benzothiazolynon hydrazone (MBTH), and achromogen such as phenol in the presence of peroxidase.

Examples of the chromogen can include phenol derivatives, anilinederivatives, toluidine derivatives, etc. Specific examples of thechromogen may include N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine,N,N-dimethylaniline, N,N-diethylaniline, 2,4-dichlorophenol,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline,N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), etc.

Further, it is also possible to use as the above-mentioned chromogen, aknown leuco-type color-developing reagent which develops a color throughthe oxidation thereof in the presence of peroxidase. Specific examplesof the color-developing reagent may include o-dianisidine, o-tolidine,3,3-diaminobenzidine, 3,3,5,5-tetramethylbenzidine,N-(carboxymethylaminocarbonyl)-4,4-bis(dimethylamino)biphenylamine (DA64), 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine(DA 67), etc.

(Other measurement methods)

In addition to the above-mentioned colorimetry method, a methodutilizing fluorescence or chemiluminescence can also be used so as tomeasure the hydrogen peroxide by using the chromogen.

In the fluorescence method (or fluolometry), it is possible to use acompound which is capable of giving fluorescence through the oxidationthereof, such as homo-vanillic acid, 4-hydroxyphenyl acetic acid,tyramine, para-cresol, and diacethyl fluorescin derivative. In thechemiluminescence method, it is possible to use peroxidase, potassiumferricyanide, hemin, etc., as a catalyst, and to use luminol, lucigenin,isoluminol, pyrogallol, etc., as a substrate.

Further, in the above-mentioned measurement of hydrogen peroxide, it isalso possible to use a system in which catalase is reacted therewith inthe presence of an alcohol (such as methanol) and the resultant aldehydeis treated by haunch reaction or the above-mentioned condensationreaction using MBTH so as to develop a color. It is possible that thethus produced aldehyde is conjugated with aldehyde dehydrogenase and theresultant change in NAD (NADH) is measured.

On the other hand, a known aldose reagent, such as diphenylamine, can beused so as to measure the glucosone which is an FAOD reaction productother than hydrogen peroxide.

(Measurement using electrode)

When the hydrogen peroxide as an FAOD reaction product is measured byuse of an electrode, the material of the electrode is not particularlylimited as long as the material can transfer (donate or withdraw)electrons with respect to the hydrogen peroxide. Preferred examples ofsuch a material may include platinum, gold, silver, etc. The measurementusing an electrode can be effected by a known method in the art, such asamperometry, potentiometry and coulometry.

Further, it is also possible to measure the glycated protein by causingan electron-transporting carrier to be concerned in the reaction as anintermediary between the electrode and FAOD or substrate, and measuringthe amount of oxidation-reduction electric current or quantity ofelectricity. The above-mentioned electron-transporting carrier may beany known material which is capable of transporting electrons, andspecific examples thereof may include ferrocene derivatives, quinonederivatives, etc.

Further, it is also possible to measure the glycated protein by causingan electron-transporting carrier to be concerned in the reaction as anintermediary between the electrode and the hydrogen peroxide produced bythe FAOD reaction, and measuring the amount of oxidation-reductionelectric current or quantity of electricity.

EXAMPLES

Hereinbelow, the present invention will be described in more detail withreference to Examples.

Example 1

(Primary screening of proteases)

As the primary screening of proteases, a glycated protein was fragmentedby use of each of these proteases, and thereafter the resultant productwas reacted with FAOD-G as an FAOD, and the amount of the resultanthydrogen peroxide was measured.

(1) Materials

Enzyme to be examined: proteases described in Table 1 appearinghereinafter

Substrate: Human serum albumin (trade name: Alb, mfd. by SIGMA Co.)

Human serum (serum, Bio Whittaker)

FAOD: FAOD-G (FAOD-G was isolated and purified from Gibberella fujikuroiby use of the method described in JP-A Hei-7-289253)

Chromogen: 4-amino antipyrine

N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine

Buffer: 0.1M Tris-HCl buffer (pH value 8.0)

(2) Protease Treatment

500 μl of human serum albumin or human serum, which had been prepared byusing the above 0.1M Tris-HCl buffer (pH value 8.0) so as to provide aconcentration thereof of 5% was mixed with 500 μl of each of proteaseswhich had been prepared by use of a buffer having an optimum pH valuefor each protease so as to provide a value of 10 U/ml, and the resultantmixture was incubated for 30 minutes at the optimum temperature for eachenzyme, and then was heated at about 90° C. for 5 minutes to stop theprotease reaction.

(3) FAOD Reaction

An "FAOD reaction mixture" having the following composition wasprepared.

Supernatant of protease-treated solution obtained in the above treatment(2) 400 μl

FAOD-G (3 U/ml) 10 μl

3 mM 4-amino antipyrine solution 30 μl

3 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine solution 30 μl

peroxidase solution (60 U/ml) 30 μl

0.1 M Tris-HCl buffer (pH value 8.0) 500 μl

The FAOD reaction mixture having the above composition was incubated at37° C. for 30 minutes and the absorbance thereof was measured at 555 nm.A blank for each sample, i.e. the absorbance based on a referencesolution containing no FAOD, was also measured, and the absorbance ofthe blank was subtracted from the absorbance of each sample obtained bythe above step thereby to obtain the actual absorbance data.

According to the above-mentioned steps, the amount of hydrogen peroxideproduced by the reaction of the each protease-treated product and theFAOD was determined by use of the absorbance thereof, and in order toevaluate the usefulness of each protease, each measured value wasconverted into relative activity as compared with the standard value(100%) which was the absorbance obtained by the Protease XIV treatment.

The results obtained by the above-mentioned measurements are inclusivelyshown in the following Tables 1 and Table 2.

                  TABLE 1                                                         ______________________________________                                        Middle-temperature range proteases                                                                                relative                                       activity (%)                                                             Temperature    Protease           Alb  Serum                                  ______________________________________                                        25° C.                                                                          1.    Aminopeptidase     10   34                                        2. Aminopeptidase I 6 21                                                      3. Carboxypeptidase B  59                                                     4. Carboxypeptidase Y 2 115                                                   5. Cathepsin B (bovine)  44                                                   6. Leucine aminopeptidase (cytosol) 56 42                                     7. Papain  21                                                                 8. Protenase A 9 6                                                            9. Trypsin  5                                                                 10. TPCK Trypsin  5                                                           11. Chymotrypsin  6                                                          30° C. 12. Lysylendopeptidase 4 11                                      13. Subtilisin A 13 47                                                        14. Protease (type XXI) 9 7                                                  37° C. 15. Achromopeptidase  21                                         16. Lysine aminopeptidase 8 25                                                17. Pronase E 99 58                                                           18. Protease 86 39                                                            19. Protease (type XIV) 100 100                                               20. Protease (type XVII)  24                                                  21. Protenase 5 91                                                         ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        High-temperature range protease                                                                                relative                                         activity (%)                                                              Temperature                                                                              Protease          Alb    Serum                                     ______________________________________                                                   Protease (type XIV)                                                                             100    100                                         50° C. 22. Human liver cathepsin B                                      23. Pronase 65 86                                                             24. Protease A 90                                                             25. Protease M 12                                                             26. Protease N 22 50                                                          27. Protease P 51 74                                                          28. Papain W-40 11 34                                                         29. Sumizyme LP50 17 32                                                       30. Sumizyme LPL  26                                                          31. Sumizyme MP 150 136                                                       32. Sumizyme FP  18                                                           33. Protin PC10 25                                                           60° C. 34. Penicilloprotenase                                           35. Protease S 138 74                                                         36. Protin A 37 91                                                            37. Protin FA 39 185                                                          38. Thermoase 99 20                                                           39. Bromelain F 35 69                                                         40. Proleser G 32 44                                                          41. Sumizyme AP 39 122                                                     ______________________________________                                    

Based on these results of the primary screening (Tables 1 and 2), tenkinds of proteases were selected since they mainly provided highabsorbance for both human serum albumin (HSA) and human serumsubstrates.

Example 2

(Secondary screening of proteases)

In this Example, with regard to the above-mentioned FAOD-S and FAOD-G asthe FAOD, the ten kinds of proteases selected in Example 1 weresubjected to a secondary screening. The FAOD-S used herein was isolatedand purified by the method described in JP-A Hei-7-289253.

In the secondary screening, measurement was performed in the same manneras in Example 1, except that the amount of the FAOD-S used in onemeasurement was changed to 0.06 U (6 U/ml, 10 μl).

The results obtained in the secondary screening are shown in thefollowing Table 3.

                  TABLE 3                                                         ______________________________________                                                      Alb           Serum                                                           FAOD activity (ΔOD 555)                                   Protease      S       G         S     G                                       ______________________________________                                        Protease XIV  0.1350  0.1100    0.0570                                                                              0.0428                                    Leucine aminopeptidase 0.0037 0.0025 0.0011 0.0191                            Trypsin 0.0064 0.0143 0.0065 0.0008                                           Protease 0.0962 0.0756 0.0481 0.0318                                          Protease 0.1273 0.0940 0.0520 0.0274                                          Protenase K 0.0496 0.0501 0.0217 0.0216                                       Protease S 0.0681 0.0210 0.0161 0.0135                                        Sumizyme AP 0.0110 ND ND ND                                                   Sumizyme MP 0.1477 0.1205 0.0553 0.0464                                       Protin FA 0.0170 ND 0.0284 0.0725                                           ______________________________________                                    

As shown in the above Table 3, it was found that both of the FAOD-S andthe FAOD-G had a similar tendency with respect to each of the proteases.Accordingly, as the most preferred protease too be used for measuringthe glycated albumin in combination with the FAOD, there were selectedSumizyme MP and Protease XIV which provided high measurement values withrespect to both of the human serum albumin (HSA) and human serumsubstrates.

Example 3

(Measurement of glycated albumin using Sumizyme MP and FAOD-G or FAOD-S)

Human serum albumin was diluted with a buffer so as to provideconcentrations of 0, 5, 10, 15, 20, 25 and 30 mg/ml, thereby to preparesamples to be measured. Then, in the same manner as in Example 2, thesamples were digested or fragmented by use of Sumizyme MP, then theFAOD-S or FAOD-G was reacted with the resultant product, and the amountof hydrogen peroxide produced in this reaction was measured in terms ofthe absorbance thereof. The thus obtained results are shown in FIG. 1.

In FIG. 1, the ordinate shows the absorbance at 555 nm, and the abscissashows the concentration of albumin. From the results shown in FIG. 1, itwas confirmed that when Sumizyme MP was used as the protease, the colordevelopment based on the action of the FAOD on the above-mentionedsample showed a good proportional relationship with respect to theconcentration of the glycated albumin, and therefore Sumizyme MP was auseful protease for measuring the glycated human albumin.

Example 4

(Various investigations on protease from A. melleus having optimum pHvalue in basic range)

Investigations were conducted with respect to the following five strainsof A. melleus, i.e., A. melleus strain (deposition number: FERM BP-6277)and strains obtained from IFO (Institute for Fermentation Osaka).

A. melleus (deposition number: FERM BP-6277)

A. melleus IFO 4339

A. melleus IFO 4420

A. melleus IFO 7541

A. melleus IFO 32035

(Renaturation and culture of strains)

The above-mentioned five strains were those which had been stored in afreeze-dried condition in ampoules, respectively, and therefore theywere renaturated by using 200 μl of a designated renaturation solutionhaving the following composition, and then were inoculated into a GPYMslant having the following composition. Each of them was cultured at 30°C. for 2 days, and then it was found that all the strains were grown.The resultant slants were stored at 4° C. In the case of culturingthereof, each of the strains was subcultured into the correspondingculture medium by use of the thus obtained slants.

    ______________________________________                                        <Composition of renaturation solution>                                        ______________________________________                                               Polypepton                                                                             0.5%                                                            Yeast extract 0.3%                                                            MgSO.sub.4.7H.sub.2 O 0.1%                                                  ______________________________________                                    

(The mixture having the above composition was adjusted to a pH value of7 and then was subjected to autoclave treatment (120° C., for 20minutes)).

    ______________________________________                                        <Composition of GPYM slant>                                                   ______________________________________                                               Glucose 1.0%                                                             Pepton 0.5%                                                                   Yeast extract 0.3%                                                            Malt extract 0.3%                                                             Agar 2.0%                                                                   ______________________________________                                    

(The above-mentioned components other than agar were dissolved indistilled water, and the pH value of the resultant mixture was adjustedto 5.5-6 and the mixture was measured by using a measuring cylinder(weighed), and then agar was added to the resultant mixture and wasdissolved therein by use of a microwave oven. The resultant compositionwas divided into test tubes so as to provide an amount thereof of 8 mlin each test tube, and then was subjected to autoclave treatment (120°C., for 20 minutes). Then, the mixture was hardened while the tube wasin a slanted state.)

Thereafter, each strain was inoculated into 10 ml of a GPYM mediumhaving the following composition, and then was cultured by a shakingculture method at 30° C. for 2 days. Then, the resultant product wastransferred into 500 ml of the same medium and was further cultured for2 days. The rate of shaking was 111 rpm for both culture steps.

    ______________________________________                                        <composition to GPYM medium>                                                  ______________________________________                                               Glucose 1.0%                                                             Pepton 0.5%                                                                   Yeast extract 0.3%                                                            Malt extract 0.3%                                                           ______________________________________                                    

(The above-mentioned components were dissolved in distilled water, andthe pH value of the resultant mixture was adjusted to 5.5-6, and wasthen subjected to autoclave treatment (120° C., for 20 minutes)).

As a result of the above culturing, each of the strains showed goodgrowth. Then, bacterial cells were collected or harvested by filtration,and the cells were weighed (wet weight). The thus obtained results areshown below.

    ______________________________________                                        <Cell weight (wet weight) of A. melleus>                                      ______________________________________                                        A. melleus  FERM BP-6277                                                                         20.6 g                                                       A. melleus  IFO 4339  20.7 g                                                  A. melleus  IFO 4420  11.1 g                                                  A. melleus  IFO 7541  8.5 g                                                   A. melleus  IFO 32035 16.3 g                                                ______________________________________                                    

The Sumizyme MP-like protease was assumed to be an extracellular enzyme,and therefore the culture supernatant obtained above was used for thesubsequent investigation. The collected cells were stored at -20° C.

(Partial purification of culture supernatant)

100 ml of the culture supernatant after the collection of the cellsobtained by the above-mentioned step was concentrated and partiallypurified by precipitation using ammonium sulfate. Since Sumizyme MP tobe selected by screening was stable in the vicinity of neutral pH valueand the pH value of the cultured medium at the end of culture wasapproximately 4.6-5, the pH value was maintained at 6-6.5 during theprecipitation using ammonium sulfate. The ammonium sulfate was addedunder cooling in an ice bath, and the resultant mixture was stirred at4° C. for 1 hour after the addition of ammonium sulfate. After thestirring, the resultant mixture was centrifuged at 10000 rpm at 4° C.for 40 minutes so as to collect the resultant precipitate, and theprecipitate was dissolved in a minimum amount of distilled water and wassubjected to dialysis at 4° C. over night. Distilled water was used asthe external solution for dialysis.

After the dialysis, the resultant internal fluid was centrifuged in amicro-tube (4° C., 12,000 rpm, 20 minutes). After the centrifugation,the resultant supernatant was collected and the volume thereof wasmeasured. The thus obtained results are shown below.

    ______________________________________                                        <pH value of culture supernatant and pH value of mixture                        at the time of completion of addition of ammonium sulfate>                  ______________________________________                                        A. melleus FERM BP-6277                                                                           pH 4.8 → pH 6.264                                    A. melleus IFO 4339 pH 5 → pH 6.4                                      A. melleus IFO 4420 pH 5.068 → pH 6.008                                A. melleus IFO 7541 pH 4.650 → pH 6.030                                A. melleus IFO 32035 pH 4.967 → pH 6.000                             ______________________________________                                    

    ______________________________________                                        <Volume of concentrated culture supernatant after dialysis>                   ______________________________________                                        A. melleus FERM BP-6277                                                                          1.6 ml                                                       A. melleus IFO 4339 3 ml                                                      A. melleus IFO 4420 2 ml                                                      A. melleus IFO 7541 3.8 ml                                                    A. melleus IFO 32035 2.2 ml                                                 ______________________________________                                    

As shown by the above results, it was found that the volume of theconcentrated culture supernatant after the dialysis was 1.6-3.8 ml. Ascompared with the volume (100 ml) thereof before the addition of theammonium sulfate, the ratio of concentration was several tens times.

(Measurement of protease activity)

The protease activity of each of the concentrated culture supernatants(five kinds of proteases) obtained by the above-mentioned step wasmeasured. At the time of the measurement of the protease activity, 20 μlof the above concentrated culture supernatant, 20 μl of 5 wt. % of HSA(pH 8) as a substrate, and 60 μl of 0.1 M Tris-HCl buffer (pH 8.0) weremixed and the resultant mixture was then subjected to reaction at 37° C.for 30 minutes. Then, 100 μl of 0.6 M trichloroacetic acid (TCA) wasadded, and the resultant mixture was left standing for 15 minutes ormore under cooling in an ice bath and thereafter was subjected tocentrifugation at 4° C. at 12000 rpm for 10 minutes so as to isolateprotein (TCA precipitation).

To 25 μl of the supernatant obtained by the above-mentioned TCAprecipitation, there were added 125 μl of Reagent-A and 1000 μl ofReagent-B of Bio-Rad DC Protein assay kit (mfd. by Bio-Rad Co.), andleft standing for 15 minutes at room temperature. Then, the absorbanceof the resultant product was measured at 750 nm so as to measure freeamino acid in the above supernatant. In order to avoid counting theamino acid which had originally been present in the culture medium asthe protease activity at the time of the above measurement of the freeamino acid, a sample which had been subjected to the protease reactionfor 0 min, was treated in the same manner as in the treatment of othersamples, and the resultant sample was used as a blank reference.

As a result of the above measurement, all the five kinds of theconcentrated cell supernatants showed clear color development after thereaction for 30 minutes, and the presence of protease activity wasconfirmed therein.

Further, the thus obtained results were compared with the amount of freeamino acid cleaved by Sumizyme MP which had been prepared so as toprovide an activity of 1 mg/ml (260 U/ml), whereby the activity of eachprotease with respect to HSA was roughly calculated. The thus obtainedresults are shown below.

    ______________________________________                                        <Protease activity (as compared with Sumizyme MP, 1 mg/ml,                      260 U/ml)                                                                   ______________________________________                                        A. melleus FERM BP-6277                                                                       0.2087 mg/ml  54.5 U/ml                                         A. melleus IFO 4339 0.1252 mg/ml 32.5 U/ml                                    A. melleus IFO 4420 0.5144 mg/ml 113.5 U/ml                                   A. melleus IFO 7541 0.0953 mg/ml 25.0 U/ml                                    A. melleus IFO 32035 0.3298 mg/ml 85.5 U/ml                                 ______________________________________                                    

(Determination of optimum pH of protease)

The optimum pH value of a protease was measured by changing the pH valueof the buffer constituting the reactant solution for measuring theactivity to be used in the above "protease activity measurement."

With respect to the pH range which could not boon covered with the 0.1 MTris-HCl buffer, 0.1 M potassium phosphate buffer and 0.1 Mglycine--NaOH buffer were used. The temperature for the reaction was setto 37° C. The protease activity was measured at a pH range from 4 to 13.The pH value used herein were set at intervals of 0.5 with respect tothe pH range of 7-10, and the pH values were set at intervals of 1.0with respect to the other pH range.

As a result, it was confirmed that each of the five kinds of enzymes hadits optimum pH value within a pH range of 8-9 which was substantiallythe same as that of Sumizyme MP. The pH dependency of the protease fromA. melleus FERM BP-6277 is shown in the graph of FIG. 2. In this graph,the enzyme activity is shown in terms of relative activity based on theactivity in the above-mentioned optimum pH value as a standard (activityin the optimum pH value is treated as "100").

Further, a peak of the activity was also observed in the acidic range.The activity of the "acid protease" was equal to or more than that of aweak-alkali protease. If these proteases were extracellular enzymes, anda protease having the highest activity at the pH value of the culturemedium was secreted in a large amount, this situation was expected tooccur with high possibility, since the pH value of the medium waslargely shifted to the acidic range at the end of the culturing.

Various characteristics of the acid proteases from A. melleus were alsoconfirmed in Examples appearing hereinafter.

(Determination of optimum temperature of protease)

The optimum temperature was measured at a fixed pH value of the bufferof 8.5 while the temperature was changed from 15 to 70° C. at intervalsof 5° C. The activity thereof was measured in the same manner as in theprotease activity measurement after it was subjected to a digestionreaction for 30 minutes.

As a result of the above measurement, it was found that each of the fivekinds of enzymes had an optimum temperature at 45° C. or 50° C. Thetemperature dependency of the protease from A. melleus FERM BP-6277 isshown in the graph of FIG. 3. In this graph, the enzyme activity isshown in terms of relative activity value based on the activity in theoptimum temperature as a standard (activity in the optimum temperatureis treated as "100"). The above-mentioned characteristic is very similarto that of "Sumizyme MP" (which practically shows its action in therange of 45-50° C. and has its optimum temperature at 50° C.).

(Estimation of molecular weight of protease from A. melleus FERMBP-6277)

Then, the protease from A. melleus FERM BP-6277, the activity of whichon a synthetic peptide was similar to that of "Sumizyem MP" wassubjected to GPC (gel filtration chromatography). Superdex 200 pg 16/60was used as a column and each of the resultant fractions was collectedat intervals of 30 seconds. By measuring the protease activity of eachfraction, the molecular weight of the protease was estimated by use ofthe time corresponding to the elution of the fraction.

    ______________________________________                                        <Conditions of GPC>                                                           ______________________________________                                        Column:       Supordex 200 pg 16/60 (Pharmacia Co.)                             Eluent: 20 mM Tris-HCl buffer                                                 Flow rate: 0.5 ml/min                                                         Temperature: Room temperature                                                 Detector: UV (220 nm)                                                         Injected volume: 50 μl                                                   ______________________________________                                    

In the measurement of the protease activity, in consideration of thedilution ratio, etc., of the protease during the GPC process, 80 μl ofeach fraction was added to 20 μl of 0.5 wt. % HSA, and subjected toreaction at 37° C. overnight. The procedure after the addition of TCAwas effected in accordance with the above-mentioned "protease activitymeasurement."

As a result, among the respective fractions obtained by the GPC, thefraction Nos. 41-43 showed a high activity, and therefore the molecularweight of the protease from this strain was presumed to be 18382-22130.

(Measurement of glycation ratio for HSA using Sumizyme MP)

Then, the measurement of glycation ratio for HSA was tried. First ofall, the glycation ratio was measured by using Sumizyme MP. Each of HSAs(SlGMA Co., Albumin Human Fraction V) having the glycation ratios of11.7, 22.5 and 26.0% (the glycation ratio was measured by use of KDKGAA-2000 (HPLC method) mfd. by Kyoto Daiichi Kagaku Co., Ltd.) wasdissolved so as to provide a concentration of 5% (pH value 8). The HSAhaving a glycation ratio of 11.7% was mixed with an equal volume of eachof the other two kinds of HSAs so as to prepare HSAs having theglycation ratios of 17.1% and 18.85% for convenience.

To 200 μl of 5 wt. % of HSA, 100 μl of 0.1 M Tris-HCl buffer and 100 μlof Sumizyme MP (10 mg/ml) were added, and then the resultant mixture wassubjected to protease reaction at 37° C. for 4 hours. Thereafter, by useof the FAOD-S (0.5602 U/ml), the activity thereof was confirmed by useof the following 4AA-TOOS color-developing system.

    ______________________________________                                        <Composition of FAOD reaction mixture>                                        ______________________________________                                        Solution after protease reaction                                                                  400 μl                                                   0.1 M Tris-HCl buffer (pH 8.0) 410 μl                                      3 mM 4-amino antipyrine 30 μl                                              3 mM TOOS 30 μ1                                                            60 U/ml POD 30 μl                                                          0.5602 U/ml FAOD-S 100 μl                                                ______________________________________                                    

As a result of the above-mentioned measurement, as shown in the graph ofFIG. 4, when the above measuring system using "Sumizyme MP"/FAOD-S wasused, it is confirmed that the glycation ratio of HSA was increasedalong with an increase in the quantity of color development. Further,there was obtained a positive correlation between the glycation ratioand the resultant absorbance, and therefore it was found that the methodusing this measurement system was useful for measuring the glycationratio of HSA.

(Measurement of glycation ratio of HSA using protease from A. melleusFERM BP-6277)

Then, with respect to the protease from A. melleus FERM BP-6277 whichhad been used for the above-mentioned GPC measurement of glycation ratioof HSA, experiments on the glycation ratio measurement for HSA wereconducted in the same manner as in that using "Sumizyme MP." However,since it had been found that the protease activity was low, the reactionwas conducted by adding 200 μl of the protease solution to 200 μl of 5wt. % of HSA, and the reaction time was 5 hours.

In the same manner as in the case using "Sumizyme MP", color developmentwas confirmed by use of the FAOD-S. As a result, as shown in the graphof FIG. 5, a positive correlation was obtained as in the investigationusing Sumizyme MP. Therefore, it was confirmed that the protease from A.melleus FERM BP-6277 was useful for the measurement of the glycationratio of HSA similarly as in the case of "Sumizyme MP."

The graph in FIG. 5 does not cross with the origin. According to thepresent inventors' knowledge, it was presumed that an impurity in theprotease and FAOD-S or a component of HSA were subjected to reaction andtherefore they caused pseudo-color development. It was presumed that thegraph approached the origin when the purity of the enzyme was improved.

Example 5

(Separation of protease having optimum pH in acidic range)

In this Example, A. melleus KDK 3001 (deposition number: FERM BP-6277)which was stored in Kyoto Daiichi Kagaku Co., Ltd., and four strains ofA. melleus (A. melleus IFO 4339, A. melleus IFO 4220, A. melleus IFO7541 and A. melleus IFO 32035) obtained from IFO (incorporatedfoundation: Research Institute for fermentation) were used as A.melleus.

Since the above-mentioned four strains obtained from IFO were stored ina freeze-dried state in ampoules, respectively, a renaturation solutionwas prepared in accordance with the designation of the supplier. Each ofthe four strains was renatured by use of 200 μl of the renaturationsolution, and then was inoculated into a GPYM slant. After the thusobtained slant was cultured at 30° C. for 2 days, it was observed thatall of the strains were grown. The composition of the GPYM slant usedherein was as follows:

    ______________________________________                                               Glucose 1.0%                                                             Pepton 0.5%                                                                   Yeast extract 0.3%                                                            Malt extract 0.3%                                                             Agar 2.0%                                                                   ______________________________________                                    

(pH 5.5-6.0, sterilized by an autoclave, 120° C., for 20 minutes)

Each of the strains (five kinds of strains in total) was inoculated into10 ml of a GPYM medium, and then was cultured at 30° C. for 2 days by ashaking culture method. Thereafter, the strain was transferred into 500ml of the same medium, and then was further cultured for 2 days. Therate of shaking was 111 rpm for each of the cultures.

As a result of the above culture, all of the strains showed good growth.Then, bacterial cells were collected by filtration, and then the cellswere weighed (wet weight). Since the protease as a target was expectedto be an extracellular enzyme, the supernatant obtained by theabove-mentioned procedure was used for the subsequent investigation. Thecomposition of the GPYM slant used herein was as follows:

    ______________________________________                                               Glucose 1.0%                                                             Pepton 0.5%                                                                   Yeast extract 0.3%                                                            Malt extract 0.3%                                                           ______________________________________                                    

(pH 5.5-6.0, sterilized by an autoclave, 120° C., for 20 minutes) 100 μlof the cultured supernatant after the collection of the bacterial cellswas concentrated and partially purified by precipitation using ammoniumsulfate. Since Sumizyme MP (commercially available) which was an enzyme(to be used in food industry) isolated and purified from A. melleus wasstable in the vicinity of neutral pH value and the pH value of thecultured medium at the end of the culturing was approximately 4.6-5, thepH value was maintained at 6-6.5 during the ammonium sulfateprecipitation.

In the ammonium sulfate precipitation, the ammonium sulfate was addedunder cooling in an ice bath, and after the addition of the ammoniumsulfate, the mixture was stirred at 4° C. for an hour. After thecompletion of the stirring, the resultant mixture was centrifuged at 4°C. at 10000 rpm for 40 minutes so as to collect precipitate, and theresultant precipitate was dissolved in a minimum volume of distilledwater and dialyzed against distilled water at 4° C. over night(semi-permeable membrane: dialysis membrane, mfd. by Sanko Jun-yaku Co.,Ltd.). After the completion of the dialysis, the resultant internalfluid was centrifuged in a centrifugal micro-tube (4° C., 12,000 rpm,for 20 minutes). After the centrifugation, the resultant supernatant wascollected.

The protease activity of each of the concentrated culture supernatantswas measured. The above-mentioned "Sumizyme MP" was a weak-alkaliprotease having an optimum pH value in the neighborhood of 8. However,according to the present inventors' experiments, it was expected that aprotease having an optimum pH value in the acidic range was presentother than the weak-alkali protease, and therefore the measurement ofthe acid protease activity was confirmed.

As a substrate for the measurement of this protease activity, 5 wt. % ofHSA (pH 5) was used. 20 μl of the concentrated culture supernatantobtained above, 20 μl of 5 wt. %-HSA and 60 μl of 0.1 M Tris-HCl buffer(pH 5) were mixed with each other, and the resultant mixture was thensubjected to reaction at 37° C. for 30 minutes. Then, to the resultantmixture, 100 μl of 0.6 M trichloroacetic acid (TCA) was added, and theresultant mixture was left standing for 15 minutes or more under coolingin an ice bath, and then was subjected to centrifugation at 4° C. at12000 rpm for 10 minutes (TCA precipitation). By use of 25 μl of theresultant supernatant from which protein had been removed by the TCAprecipitation, the amount of free amino acids in the supernatant wasmeasured by use of commercially available Bio-Rad DC Protein assay kit(mfd. by Bio-Rad Co.). The kit was used in a manner according to thesupplier's instructions. At the time of the measurement of free aminoacids, in order to avoid the effect of amino acids contained in theconcentrated culture supernatant, a sample which had been obtained bymixing the above three kinds of solutions, and immediately thereaftersubjecting the resultant mixture to the TCA precipitation was used as ablank (reference).

As a result, all of the five kinds of concentrated cultured supernatantcorresponding to the above-mentioned five kinds of strains showed clearcolor development after the above reaction for 30 minutes, and thereforethe presence of the protease activity was confirmed. Based on theconfirmation of those protease activities, each of the partiallypurified concentrated cultured supernatants was used as a proteasesolution for the subsequent investigations.

Then, among the above-mentioned five kinds of strains, the solutioncontaining the protease from A. melleus KDK 3001 strain was subjected tothe measurement of the optimum pH value thereof.

In the measurement of the optimum pH value, first of all, 5 wt. % of HSAsolutions having a pH value of 1.6 and a pH value of 8 were prepared asa substrate solution by use of 0.1 M Tris-HCl buffer, then by mixingthese two solutions, ten kinds of substrate solutions having their pHvalues in the range of pH 1. 6-pH 6.8 were prepared in total. Further,by adding hydrochloric acid to the HSA solution having a pH value of1.6, a substrate solution having a pH value of 1 was prepared.

20 μl of each of the substrate solutions the pH values of which had beenadjusted to each of the above-mentioned pH values, 20 μl of theprotease-containing solution and 60 μl of distilled water were mixedwith each other, and then the resultant mixture was subjected toreaction at 37° C. for 30 minutes. The pH value of the reaction mixturewas measured again just before the reaction (i.e. just after the mixingof three solutions), and the pH value of the reaction mixture wasmeasured again at the time of the protease reaction (just after mixingof the three solutions). As a result, it was found that the pH value ofthe reaction mixture was in the range of 1.6-6.5.

The enzyme activity was measured by removing protein by use of TCAprecipitation and measuring the amount of free amino acids in theresultant supernatant by use of Bio-Rad DC Protein assay kit in the samemanner as in the above-mentioned protease activity measurement.

As a result, it was confirmed that the acid protease from A. melleus KDK3001, as shown in the graph of FIG. 6, was one having its optimum areain the range between pH 4.7 and pH 5.6 and having a particularly highactivity at pH 5.

The optimum temperature of the acid protease from A. melleus KDK 3001was measured by fixing the pH value of the reaction mixture at pH 5 andchanging the temperature from 15 to 70° C. at intervals of 5° C. Afterthe incubation for 30 minutes, the protease activity was measured in thesame manner as that described above. As a result, as shown in the graphof FIG. 7, the protease practically functioned in the range of 35-65°C., and had a high activity in the range of 40-50° C., and therefore itwas found that the protease had its optimum temperature within thisrange. The protease showed the highest activity when the temperature was50° C.

In order to roughly measure the molecular weight of the protease, theprotease from A. melleus KDK 3001 was subject to GPC. Superdex 200 pg16/60 (mfd. by Pharmacia Co.) was used as a column and fractions werecollected at intervals of 30 seconds. In the GPC treatment, by measuringthe protease activity of each of the collected fractions, the molecularweight of the protease was presumed by use of the time corresponding tothe elution of the fraction. The measurement condition for the GPC usedherein were as follows.

    ______________________________________                                        <Measurement conditions of GPC>                                               ______________________________________                                        Column:     Superdex 200 pg 16/60 (mfd. by Pharmacia Co.)                       Eluent: 20 mM Tris-HCl buffer                                                 Elution condition: 0.5 ml/min                                                 Temperature: Room temperature                                                 Detection: UV detection (220 nm)                                              Volume of injected 50 μl                                                   sample:                                                                     ______________________________________                                    

In the measurement of the protease activity, in view of the dilution ofprotease during the GPC process, 80 μl of each fraction was added to 20μl of 0.5 wt. % HSA, and the resultant mixture was subjected to reactionat 37° C. for 24 hours, and then the protease activity thereof wasmeasured in the same manner as described above. The protease activity ofeach of the fractions obtained by the above-mentioned GPC was measuredby the same measurement method as described above, and it was estimatedthat the molecular weight of the protease from this strain was18382-22130.

Example 6

(Measurement of glycated protein by use of acid protease)

In this Example, based on the advantage that the protease obtained inExample 5 had its optimum pH value in the acidic range while the optimumpH value of the FAOD was 8, the protease activity was inactivated byadjusting the pH value to the optimum pH value of the FAOD (pH 8) afterthe protease treatment, without heating or adding an inhibitor.

By use of a solution containing the protease which had been isolated andpurified from A. melleus KDK 3001, human serum albumin (HSA: Sigma Co.,"ALUBUMIN HUMAN FRACTION V", and Bayer Co., "Albumin FrV") havingdifferent glycation ratios were subjected to the measurement. Theglycation ratios of these HSAs were 11.7%, 22.5% and 26.0%,respectively. These glycation ratios were measured in advance by use ofGAA-2000 (HPLC method) mfd. by Kyoto Daiichi Kagaku Co., Ltd.

In the subsequent measurement of the glycated HSA by using an enzymaticmethod, the above-mentioned various glycated HSAs were used after theywere adjusted to a concentration of 5 wt. % and a pH value of 5.Further, by mixing HSA samples having different glycation ratios,samples having their glycation ratios of 17.1% and 18.85% were alsoprepared. By use of these five kinds of samples having differentglycation ratios, the glycated HSA was measured by use of theprotease-containing solution obtained in Example 5.

However, since it had been found that the protease activity obtained inExample 5 was low, 200 μl of the protease-containing solution of Example5 was added to 200 μl of 5 wt. % of HSA so as to cause the reaction, andthe reaction time was 5 hours. After the reaction using this protease,the resultant color development was confirmed by use of the FAOD(FAOD-S). As the FAOD, the FAOD-S which had been prepared according toJP-A Hei-7-289253 (0.5602 U/ml) was used.

The titer of the FAOD used in this Example was measured by theabove-mentioned rate method. The composition of the reactant solution ofthe FAOD color-developing system used for the above-mentioned titerdetermination was as follows.

    ______________________________________                                        Solution after the above protease reaction                                                           400 μl                                                0.1 M Tris-HCl buffer (pH 8.0) 410 μl                                      3 mM 4-Aminoantipyrine 30 μl                                               3 mM TOOS 30 μl                                                            60 U/ml POD (peroxidase) 30 μl                                             0.5602 U/ml the FAOD-S 100 μl                                            ______________________________________                                    

After the FAOD color-developing solution having the above-mentionedcomposition was incubated at 37° C. for 30 minutes, the absorbancethereof at 555 nm was measured. With respect to each of the abovesamples, a blank (i.e. the absorbance of the reference solutioncontaining no substrate) was also measured, and the absorbance of theblank was subtracted from the absorbance of each sample obtained by theabove step, thereby to obtain the actual absorbance.

As a result of the above measurement, as shown in the graph of FIG. 8,the color-developing reaction was caused by the FAOD corresponding tothe glycation ratio of the HSA. Accordingly, it was confirmed that theacid protease obtained by this Example was sufficiently usable formeasuring a glycated protein in combination with the FAOD.

The graph in FIG. 8 does not cross with the origin. According to thepresent inventors' knowledge, it was presumed that the correspondingcolor development was attributable to an impurity in the protease andFAOD or a component of HSA used herein. It was presumed that the graphapproached the origin when the purity of the enzyme was improved.

Industrial Applicability

As described hereinabove, according to the present invention, there isprovided a protease to be used for measuring a glycated protein in asample in combination with FAOD (fructosyl amino acid oxidase).

The present invention also provides a method of measuring a glycatedprotein by causing FAOD to act on a sample containing a glycatedprotein, wherein the glycated protein is treated with a protease underan acid condition.

The present invention further provides a method of measuring a glycatedprotein by causing protease and FAOD to act on a sample containing aglycated protein, wherein a protease from Aspergillus genus is used asthe protease.

The present invention further provides a method of measuring a glycatedprotein by causing protease and FAOD to act on a sample containing aglycated protein, wherein Protease XIV is used as the protease.

In the above-mentioned method according to the present invention, aglycated protein in a living organism component can be measured withhigh sensitivity and accuracy by using a suitable protease whichexhibits a useful enzymatic action in combination with an FAOD suitablyusable for measuring glycated albumin. Accordingly, the presentinvention provides a method of measuring a glycated protein which cancontribute to the control and prevention of symptom of diabetes, andalso provides a protease which is suitably usable for such a method.

Further, according to an embodiment of the present invention using aneasily available protease, such as "Sumizyme MP" as the above-mentionedprotease, the present invention becomes applicable to clinical use morewidely.

In addition, according to an embodiment of the present invention usingan acid protease, the above-mentioned protease can be inactivated easilyand rapidly by adjusting the pH value, and therefore the presentinvention is applicable to clinical examination, etc., more widely.

What is claimed is:
 1. A protease to be used for measuring a glycatedprotein in a sample in combination with FAOD (fructosyl amino acidoxidase), which protease is obtained from the Aspergillus genus.
 2. Theprotease according to claim 1, which is from Aspergillus melleus.
 3. Amethod of measuring a glycated protein by causing FAOD to act on asample containing a glycated protein, wherein the glycated protein istreated with a protease from the Aspergillus genus under an acidcondition.
 4. A method according to claim 3, wherein the FAOD is FAOD-Sor FAOD-G.
 5. A method according to claim 3, wherein the glycatedprotein is glycated albumin.
 6. A method of measuring a glycated proteinby causing protease and FAOD to act on a sample containing a glycatedprotein, wherein a protease from Aspergillus melleus (A. melleus) isused as the protease.
 7. A method according to claim 6, wherein theprotease was a protease from A. melleus and the protease has its optimumpH value in acid range.
 8. A method according to claim 7, wherein theprotease from A. melleus is Sumizyme MP.
 9. A method according to claim6, wherein the FAOD is FAOD-S or FAOD-G.
 10. A method according to claim6, wherein the glycated protein is glycated albumin.
 11. A method ofmeasuring a glycated protein by causing protease and FAOD to act on asample containing a glycated protein, wherein Protease XIV is theprotease.
 12. A method according to claim 11, wherein the FAOD is FAOD-Sor FAOD-G.
 13. A method according to claim 11, wherein the glycatedprotein is glycated albumin.
 14. The method according to claim 3,wherein the protease is from Aspergillus melleus.
 15. A method ofmeasuring a glycated protein by causing FAOD to act on a samplecontaining a glycated protein, wherein the glycated protein is treatedwith a protease from Aspergillus genus under an alkaline condition. 16.The method according to claim 15, wherein the protease is fromAspergillus melleus.
 17. The method according to claim 15, wherein theglycated protein is glycated albumin.
 18. The method according to claim15 wherein the wherein the FAOD is FAOD-S or FAOD-G.